This R15 proposal aims at the reverse genetic characterization of the DRG gene family in Arabidopsis thaliana. DRGs represent a subfamily of G-proteins that were originally characterized as being developmentally regulated in the mouse. One focus area of the PI, Joel Stafstrom, has been to characterize genes controlling the dormancy and the breaking of dormancy of axillary buds. During an earlier screen for differentially expressed genes, Stafstrom encountered one DRG gene, DRG2, which was induced during the breaking of axiallary bud dormancy in pea. His underlying hypothesis is that DRG genes, which are highly conserved throughout the eukaryotes, play fundamental roles in the regulation of cell differentiation during plant development. In continuation of studies initiated under a previous R15 grant, the PI now proposes to complete the molecular cloning of all three Arabidopsis DRG genes. An update provided a few weeks ago showed that genomic clones for all three are now in hand. The second goal is to further elucidate the expression patterns of the DRG genes using RNA expression assays as well as promoter constructs tagged with GUS or GFP reporters in transgenic plants. The main agenda of this proposal is to then perturb the function of the DRG genes in order to determine their role during plant growth. To this end, Stafstrom will make use of gene silencing methods, screen for insertional knockout mutations, and express potentially dominant negative mutants. He will also create knockouts of two DRG homologs in yeast. Potential mutants will be characterized for developmental and physiological defects that may give clues concerning the cellular mode of action of the DRGs and their physiological role.